Russell N. Spear

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) ofAureobasidium pullulans

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.

FLUORESCENT LABELS, CONFOCAL MICROSCOPY, AND QUANTITATIVE IMAGE ANALYSIS IN STUDY OF FUNGAL BIOLOGY

Publisher Summary The chapter discusses the use of fluorescent labels, confocal microscopy, and quantitative image analysis in study of fungal biology. Another major area of recent technical advance considered in the chapter is quantitative image analysis. In theory, it allows for the rapid and relatively objective enumeration and classification of fungal propagules meeting specific descriptions. Briefly, this procedure involves the manipulation of pictorial information. Its role in biological investigation is accelerating with the advent of modestly priced computers powerful enough to handle pictorial. As a case study of the biology of a fungus in its natural habitat, it is demonstrated that Green fluorescent protein (GFP)-transformed A. pullulans may be imaged in situ by either confocal or conventional microscopy and by the use of single or multiple probes. Green fluorescent protein has proven to be a valuable aid for identifying the subcellular location of proteins and for monitoring gene expression. There are three basic requirements for GFP expression in fungi a reliable transformation system; functional transcriptional control elements to drive expression; and the appropriate GFP-encoding gene. For routine applications, conventional microscopy coupled with digital image collection and deconvolution of the image stacks offers an alternative to confocal microscopy in several aspects.

Streptomycin Application Has No Detectable Effect on Bacterial Community Structure in Apple Orchard Soil

ABSTRACT Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 μg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielous evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.

Single-Leaf Resolution of the Temporal Population Dynamics of Aureobasidium pullulans on Apple Leaves

ABSTRACT The abundance of phylloplane microorganisms typically varies over several orders of magnitude among leaves sampled concurrently. Because the methods traditionally used to sample leaves are destructive, it has remained unclear whether this high variability is due to fixed differences in habitat quality among leaves or to asynchronous temporal variation in the microbial population density on individual leaves. We developed a novel semidestructive assay to repeatedly sample the same apple leaves from orchard trees over time by removing progressively more proximal ∼1-cm-wide transverse segments. Aureobasidium pullulans densities were determined by standard leaf homogenization and plating procedures and were expressed as CFU per square centimeter of segment. The A. pullulans population densities among leaves were lognormally distributed. The variability in A. pullulans population densities among subsections of a given leaf was one-third to one-ninth the variability among whole leaves harvested concurrently. Sequential harvesting of leaf segments did not result in detectable changes in A. pullulans density on residual leaf surfaces. These findings implied that we could infer whole-leaf A. pullulans densities over time by using partial leaves. When this successive sampling regimen was applied over the course of multiple 7- to 8-day experiments, the among-leaf effects were virtually always the predominant source of variance in A. pullulans density estimates. Changes in A. pullulans density tended to be synchronous among leaves, such that the rank order of leaves arrayed with respect to A. pullulans density was largely maintained through time. Occasional periods of asynchrony were observed, but idiosyncratic changes in A. pullulans density did not contribute appreciably to variation in the distribution of populations among leaves. This suggests that persistent differences in habitat (leaf) quality are primarily responsible for the variation in A. pullulans density among leaves in nature.

The Infection Process in Pines By Gremmeniella abietina

Gremmeniella abietina infects shoots of pines through stomata numbering about 3–20 in each of two short irregular rows on the abaxial surface of bracts that subtend short shoots. The germ tube penetrates between guard cells, sparsely colonizes bract tissue, and reaches the periderm that separates bract from stem tissue by late summer or fall. The fungus crosses this periderm only after about early February and then rapidly colonizes the short shoot and surrounding cortical tissue. This invasion produces a resinous brown necrotic area of cortical parenchyma and phloem beneath the bract as the first visible symptom of infection.

Effect of Environmental Factors on the Post-Infection Behavior of Gremmeniella abietina

Conidial production by Gremmeniella abietina was enhanced by growing the fungus in 8-oz. jars containing V-8 agar and a red pine twig. Survival of G. abietina at temperatures of 12 to 34C was closely related to average temperature and duration of exposure. The fungus survived 27 months in vivo without causing extensive necrosis of the host. Symptom development was more frequent among trees placed outdoors than among trees placed in a coldroom or greenhouse.